SNAP-tag2: faster and brighter protein labeling

Stefanie Kühn; Veselin Nasufović; Jonas Wilhelm; Julian Kompa; Eline M.F. de Lange; Yin‐Hsi Lin; Cornelia Egoldt; Jonas Fischer; Artem Lennoi; Mirosław Tarnawski; Jochen Reinstein; Rifka Vlijm; Julien Hiblot; Kai Johnsson

doi:10.1038/s41589-025-01942-z

BiochemistryNature Chemical BiologyJul 3, 2025

Peer-reviewed journal article

SNAP-tag2: faster and brighter protein labeling

Stefanie Kühn iD, Veselin Nasufović iD, Jonas Wilhelm iD, Julian Kompa iD, Eline M.F. de Lange iD, Yin‐Hsi Lin iD, Cornelia Egoldt, Jonas Fischer, Artem Lennoi, Mirosław Tarnawski iD, Jochen Reinstein iD, Rifka Vlijm iD, Julien Hiblot iD, Kai Johnsson iD

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Abstract

SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here we introduce new labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 107 s-1 M-1, a 100-fold improvement over the corresponding SNAP-tag-substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a 5-fold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translates into a greatly improved performance in various live-cell (super-resolution) imaging applications.